The immunoreceptor NKG2D is normally expressed on human T cells (e.g. CD8+ T cells, γδ T cells) and NK cells. On pre-activated CD8+ cells, NKG2D functions as a synergistic co-stimulator of CD28 and TCR signalling via DAP10 association, whereas in NK cells it functions as a direct activator. Upon ligand engagement, NKG2D therefore conveys directly activating or costimulatory signals via the paired DAP10 adaptor protein, thereby promoting cancer and infectious disease immunity.
Various ligands for human NKG2D (hNKG2D) have been identified and characterized, including the major histocompatibility complex class I-related chain A and B polypeptides (MICA and MICB), the UL16-binding protein (ULBP) family, and the retinoic acid early transcript-1 (RAET1) family. MICA is frequently associated with epithelial tumors, induced by microbial infections, and aberrantly expressed in certain autoimmune disease lesions. The structure of MICA is similar to the protein fold of MHC class I, with an α 1α2 platform domain and a membrane-proximal Ig-like α3 domain (Li et al 2001 Nat. Immunol. 2:443). MICA and its close relative MICB, which also serves as a ligand for NKG2D, are both polymorphic and the polymorphism has been shown to affect the affinity for NKG2D (Steinle et al. 2001 Immunogenetics 53:279).
In the mouse, which lacks MHC class I chain (MIC) genes, a family of proteins structurally related to ULBP, the retinoic acid early (RAE-1) molecules function as ligands for NKG2D. RAE-1 expression has been shown to be induced by carcinogens and to stimulate antitumor activities of T cells. Murine NKG2D, however, recognizes human MICA polypeptides (Wiemann (2005) J. Immunol. 175:820-829).
The role MICA in cancer biology has been complicated by the fact that MICA is released as a soluble form from the cell surface of tumor cells (e.g., *019 allele) and on the surface of exosomes (*08 allele) (Ashiru et al (2010) Cancer Res. 70(2):481-489)). Soluble MICA (sMICA) can be detected for example at high levels in sera of patients with gastrointestinal malignancies (Salih et al, 2002 J. Immunol. 169: 4098). The MMPs ADAM10 and ADAM17, as well as the disulfide isomerase Erp5, have been reported to have a role in cleavage and shedding of MICA (Waldhauer (2008) Cancer Research 68 (15) 6368-76; Kaiser et al (2007) Nature; and Salih (2002) J. Immunol 169: 4098-4102). Membrane bound MICA has been reported to downmodulate the expression of NKG2D on NK and/or T cells (Von Lilienfeld-Toal et al. (2010) Cancer Immunol. Immunother.). Notably, Wiemann (2005), supra, examined MICA Tg mice and concluded that down-regulation of surface NKG2D on nontransgenic splenocytes was most pronounced after cocultivation with splenocytes from MICA transgenic mice in vitro, and only marginally following treatment with sera from H2Kb-MICA mice, whereas incubation with control cells and sera from nontgLM, respectively, had no effect and that overall data suggest that reduced surface NKG2D on H2-K-MICA NK cells results in NKG2D dysfunction and that NKG2D downregulation is primarily caused by a persistent exposure to cellbound MICA in vivo.
Reports have also emerged that NKG2D on NK cells is downregulated by sMICA (Groh et al. (2002) Nature; Arreygue-Garcia (2008) BMC; Jinushi et al. (2005) J. Hepatol.), leading to less reactive NK cells. This rationale may have emerged because similar systems have been observed among other protein families such as the Ig-like and the TNF superfamily have been shown to be released as a soluble form and that release of the molecules affects cell-cell interactions by reduction of ligand densities and modulates NK cells bearing the respective receptor (Salih 2002). Consequently, attempts to generate anti-MICA antibodies have focused on development of antibodies that inhibit MICA shedding.
It has also been reported that expression of NKG2D ligands MICA and MICB on healthy cells can break the balance between immune activation and tolerance, and trigger autoimmunity. Genetic linkage studies have shown that some MICA alleles are positively associated with type 1 diabetes, and development of disease in prediabetic NOD mice expressing Rae1 on their islet cells can be completely prevented by treatment with NKG2D-blocking mAbs, which reduce expansion and function of autoreactive CD8+ T cells. MICA and MICB molecules are also dramatically upregulated in RA synoviocytes and activate the T cells in an NKG2D-dependent manner. Moreover, rheumatoid arthritis patients have been reported to have high levels of IL-15 and TNF-α in the sera and inflamed joints which induce expression of NKG2D on CD4+CD28− subset of T cells. In Celiac disease, massive infiltration of intraepithelial NKG2D+ CD8+ cd T lymphocytes in the gut has been reported, and MIC proteins become strongly expressed on the surface of epithelial cells in patients with active disease. In inflammatory bowel disorders, increased levels of MIC expression were found on intestinal epithelial cells and it the number of intestinal epithelial CD4+ T cells expressing NKG2D was found to correlate with intestinal inflammation.
Approaches to date to treat inflammation based on the NKG2D system have focused on blockade of NKG2D itself rather than its ligands (Ogasawara et al. (2004) Immunity 20(6):757-767; Andersson et al (2011) Arthritis. Rheum. 63(9):2617-2629; Steigerwald et al (2009) MAbs 1(2):115-127. One possibility is that this focus on NKG2D rather than its ligand is due to the perceived difficulty of targeting the NKG2D ligand system which includes a variety of ligands and in some cases a large number of alleles.
For MICA and MICB, there are over 50 MICA alleles and at least 13 MICB alleles recognized. There is only 43% amino acid identity across the MIC polypeptides in the α1α2 domain (the domain involved in the NKG2D interface), and 80% of the amino acid substitutions are non-conservative (Steinle et al. (2001) Immunogenetics 53: 279-287; Steinle et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:12510-12515), suggesting that it will be unlikely to obtain antibodies that are effective for a majority of individuals in a population. Additionally, the methionine/valine bimorphism at position 129 in MICA determines differences in NKG2D binding, and although the side chain of residue 129 is partially buried and forms hydrophobic interactions with glutamine 136, alanine 139 and methionine 140 in the first α2 helical stretch, it may be associated with a difference in conformation in this domain in comparison with valine 129 forms of MICA (Steinle et al (2001) Immunogenetics 53: 279-287).
In conclusion, there is a need for new approaches to target MICA with therapeutic agents.